The object of this proposal is to study the regulatory mechanisms underlying transcription of human cellular genes using membrane-free, cell-free systems containing human KB cell RNA polymerases and necessary factors. Human adenovirus is chosen as the model system for these studies. Most genes of adenovirus are probably transcribed by RNA polymerase II and two genes by RNA polymerase III. However, it is not clear how the polymerases interact with the template, whether factors other than purified RNA polymerases are required for faithful transcription and what are the primary transcripts. Recently I have successfully developed the first eukaryotic DNA-directed membrane-free, cell-free system from human KB cells for this purpose (Wu, G.J. 1978 Proc. Nat. Acad. Sci. USA, 75 2175-179.